The entry of thyroid hormone (TH) into cells is a critical, but poorly understood process. Alterations of TH entry into cells may contribute to changes in thyroid hormone metabolism during fasting, in severely ill patients (NTI), in hypothyroidism, and as a consequence of drugs. Altered entry of TH into cells has been described in patients with abnormal TH serum binding proteins and as a primary inherited defect producing generalized resistance to TH. The long term goal of these studies is to delineate the mechanisms and regulation of the entry of thyroid hormone into human cells and to evaluate its contributions to clinical problems. Cultured human hepatocytes, Hep G2, will be utilized as they retain many properties of normal human liver cells and allow experimental conditions to be controlled. Incubation of these cells with radioiodine labeled T3 and T4 with unlabeled TH and inactive metabolites will establish if human liver cells have saturable uptake systems for TH that are functional at physiologic concentrations, and specific for active TH. Incubation with varying concentrations of free T3 and T4 and purified albumin, thyroxine binding prealbumin (TBPA), and thyroxine binding globulin (TBG) will indicate if these proteins facilitate uptake of TH. Incubation of cells with radioiodine labeled albumin, TBPA, and TBG will determine whether these serum proteins are specifically bound to human liver cells. Uptake of tracer TH from serum of subjects with abnormal serum TH binding proteins will evaluate whether these proteins alter the cellular uptake. Incubation of TH with furosemide and free fatty acids will determine whether these postulated inhibitors of TH binding to serum proteins in NTI also interfere with cellular entry of TH. Treatment of Hep G2 cells with drugs which block cellular ATP generation will indicate whether the uptake of thyroid hormone into human cells is energy dependent. Pre-treatment with drugs which inhibit endocytosis will indicate whether this process is important in the uptake of TH by human cells. Membrane preparations obtained from the Hep G2 cells will be combined with varying concentrations of labeled and unlabeled T3 and T4 to detect specific membrane receptors. Incubation of labeled TH with ipodate, amiodarone and propranolol will evaluate if they alter cellular entry of TH by competing for uptake, inhibiting deiodination, or decreasing cellular ATP content. The effect of long term treatment of Hep G2 cells with hypothyroid serum and serum with excess TH will establish whether thyroid hormone status may alter the entry of TH into human cells.